Low molecular weight GTP/GDP binding GTPases such as Ras and Rho transduce mitogenic and survival signals from cell surface receptors to the nucleus. See, for example, McCormick, F. Nature 363: 15–16, (1993); Campbell, S. L., et al., Oncogene 17: 1395–1413, (1998); and Zohn, I. M., et al. Oncogene 17: 1415–1438, (1998). For example, platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) stimulate cell proliferation and survival by activating their receptor tyrosine kinases, which recruit nucleotide exchange factors that activate Ras by converting it to its GTP-bound state. Once activated, Ras triggers a complex set of signal transduction pathways. These include the phosphatidylinositol-3-kinase/Akt pathway believed to be critical for cell survival, and the Raf/Mek/Erk kinase cascade that has been implicated in cell proliferation. In addition to its involvement in regulating proliferation and survival, Ras also plays a pivotal role in malignant transformation. In about 30% of all human cancers, Ras is mutated to a GTPase-deficient form that leads to constitutive activation of the above signaling pathways, uncontrolled proliferation, and survival of human tumors. Barbacid, M. Annu. Rev. Biochem. 56: 779–827, (1987); and Bos, J. L. Cancer Res. 49: 4682–4689, (1989). Family members (closely related) to Ras, such as RhoA and Rac1, have also been shown to be intimately involved in proliferation and transformation. For example, both RhoA and Rac1 are required for the G1 to S phase transition during the cell division cycle. Olson, M. F., et al., A. Science 269: 1270–1272, (1995). Furthermore, GTP-locked RhoA and Rac1 are transforming, and dominant negative forms of these GTPases inhibit Ras-induced malignant transformation. Khosravi-Far, R., et al., Mol. Cell. Biol. 15: 6443–6453, (1995) and Qiu, R.Get al., Nature 374: 457–459 (1995). Unlike RhoA and Rac1, less is known about the involvement of RhoB GTPase in proliferation and transformation. There are several features that distinguish RhoB from other Rho proteins. Firstly, its cellular localization within early endosomes and the pre-lysosomal compartment is different from the localization of other members. Mellow, H. et al., J. Biol. Chem. 273: 4811–4814, (1998). Secondly, RhoB is an immediate early response gene that is induced by PDGF, transforming growth factor-α, the non-receptor tyrosine kinase v-src, and ultraviolet irradiation. Jahner, D. et al., Mol. Cell. Biol. 11: 3682–3690, (1991), and Fritz, G., et al., J. Biol. Chem. 270: 25172–25177, (1995). However, these studies were mostly performed using fibroblasts, and whether RhoB is also an immediate early response gene in human cancer cells of epithelial origin is not known. Third and finally, RhoB mRNA and RhoB protein levels turn over much more rapidly (with half-lives of 20 and 120 min, respectively) than other GTPases, which typically have half lives on the order of 24 hrs. Therefore, although RhoA and RhoB share 90% amino acid sequence homology, their physiological functions are predicted to prefers a leucine. Ras proteins (e.g. H-, K- and N-Ras) are farnesylated, whereas RhoA and Rac1 are geranylgeranylated. Although RhoB has a C-terminal leucine, which would be predicted to dictate only geranylgeranylation, it is both farnesylated and geranylgeranylated in cells. Because Ras is constitutively activated in 30% of human cancers and Ras farnesylation is required for its malignant transforming activity, Lebowitz, P. F., et al., J. Biol. Chem. 272: 15591–15594, (1997), FTase inhibitors (FTIs) were designed as novel anticancer drugs. FTIs have shown impressive antitumor activity and lack of toxicity in preclinical models and are presently in various human clinical trial phases. Sebti, S. M., et al., Pharmacol. Ther. 74: 103–114, (1997); Gibbs, J. B., et al., Annu. Rev. Pharmacol. Toxicol. 37: 143–166, (1997); and Cox, A. D., et al., Biochim. Biophys. Acta 1333: F51–F71, (1997). Although FTIs were initially hypothesized to inhibit tumor growth by targeting Ras, recent evidence suggests that other farnesylated proteins may be involve, Lebowitz, P. F., et al., Oncogene 17: 1439–1445, (1998).
RhoB has been suggested as a potential candidate for several reasons. Firstly it is a substrate for FTase and FTIs inhibit its farnesylation, resulting in decreased RhoB-F and increased RhoB-GG. Secondly, RhoB's short half life more closely resembles the kinetics of FTIs reversal of transformation than Ras. Third, a RhoB/RhoA chimeric protein that is exclusively geranylgeranylated is growth inhibitory. Thereby, a myristylated form of RhoB which is not prenylated prevents FTIs from inhibiting Ras transformation. However, the biochemical properties of myristylated RhoB are not the same as wild type RhoB making it difficult to interpret the data. Furthermore, RhoB has been shown to be farnesylated by FTase as well as by GGTase I. Fourth and final, most of the studies carried out so far used murine fibroblasts. Therefore, although there is some evidence suggesting RhoB's involvement in FTIs antitumor activity, direct evidence implicating RhoB in FTIs mechanism of action in human tumors is lacking.
A novel function for RhoB(WT) as a potent inhibitor of malignant transformation and a suppressor of human tumor growth is disclosed herein. Furthermore, both RhoB-F and RhoB-GG induce apoptosis, inhibit oncogenic signaling and suppress transformation in vitro and in vivo. These findings demonstrate the tumor suppressing activity of RhoB, and strongly suggest that, contrary to prior suggestions, RhoB-F is not a target for FTIs in human cancer cells.
While the GTPase RhoA has been shown to promote proliferation and malignant transformation, RhoB's involvement in these processes is not well understood. RhoB is described herein as a potent suppressor of transformation and human tumor growth in nude mice. In several human cancer cell lines, RhoA promotes focus formation whereas RhoB is as potent as the tumor suppressor p53 at inhibiting transformation in this assay. It is demonstrated herein that both RhoB-F and RhoB-GG inhibit anchorage-dependent and -independent growth, induce apoptosis, inhibit constitutive activation of Erk and IGF-1 stimulation of Akt and suppress tumor growth in nude mice. The data demonstrate that RhoB is a potent suppressor of human tumor growth and that RhoB-F is not a target for farnesyltransferase inhibitors. RhoB therefore provides a novel target for therapeutic intervention in the treatment and prevention of cancer as disclosed herein.